Background Mutations in ATP1A2 gene encoding the Na,K-ATPase alpha(2) isoform are associated with familial hemiplegic migraine type 2. Migraine with aura is a known risk factor for heart disease. The Na,K-ATPase is important for cardiac function, but its role for heart disease remains unknown. We hypothesized that ATP1A2 is a susceptibility gene for heart disease and aimed to assess the underlying disease mechanism. Methods and Results Mice heterozygous for the familial hemiplegic migraine type 2-associated G301R mutation in the Atp1a2 gene (alpha(+/G301R)(2) mice) and matching wild-type controls were compared. Reduced expression of the Na,K-ATPase alpha(2) isoform and increased expression of the alpha(1) isoform were observed in hearts from alpha(+/G301R)(2) mice (Western blot). Left ventricular dilation and reduced ejection fraction were shown in hearts from 8-month-old alpha(+/G301R)(2) mice (cardiac magnetic resonance imaging), and this was associated with reduced nocturnal blood pressure (radiotelemetry). Cardiac function and blood pressure of 3-month-old alpha(+/G301R)(2) mice were similar to wild-type mice. Amplified Na,K-ATPase-dependent Src kinase/Ras/Erk1/2 (p44/42 mitogen-activated protein kinase) signaling was observed in hearts from 8-month-old alpha(+/G301R)(2) mice, and this was associated with mitochondrial uncoupling (respirometry), increased oxidative stress (malondialdehyde measurements), and a heart failure-associated metabolic shift (hyperpolarized magnetic resonance). Mitochondrial membrane potential (5,5 ',6,6 '-tetrachloro-1,1 ',3,3 '-tetraethylbenzimidazolocarbocyanine iodide dye assay) and mitochondrial ultrastructure (transmission electron microscopy) were similar between the groups. Proteomics of heart tissue further suggested amplified Src/Ras/Erk1/2 signaling and increased oxidative stress and provided the molecular basis for systolic dysfunction in 8-month-old alpha(+/G301R)(2) mice. Conclusions Our findings suggest that ATP1A2 mutation leads to disturbed cardiac metabolism and reduced cardiac function mediated via Na,K-ATPase-dependent reactive oxygen species signaling through the Src/Ras/Erk1/2 pathway.